MEMBRANE BOUND DETOXICATION ENZYMES

潜在有毒异生物质的代谢与解毒 分子依赖于几个膜结合的 酶包括细胞色素P-450、微粒体环氧化物 水解酶和UDP-葡糖醛酸基转移酶。 环氧化物水解酶 催化水与环氧化物的反式加成， 由细胞色素P-450对不饱和脂肪酸的作用而产生。 芳香烃. UDP-葡萄糖醛酸转移酶催化 从UDP-葡糖醛酸盐到疏水分子的葡糖醛酸基 带有亲核官能团如-OH、-SH、-NH 2和- 哦。 这些反应的最终结果是提高溶解度， 母体分子的运输和排泄。 具体目标为 拟议的项目期间集中在三个一般领域， 调查，包括改善表达系统， 蛋白质，阐明酶的作用机制 并通过X射线对酶的结构进行了初步研究 结晶学和生物化学技术。 的具体目标 关于微粒体环氧化物水解酶的项目是：（I） 通过前稳态和稳态来阐明动力学机制， （二）化学反应机理的定义; 通过定点诱变的催化和酯的表征 （iii）评价潜在的基于机制的抑制剂 和（iv）产生X射线衍射质量的单 晶体 调查UDP的具体目的- 葡糖醛酸基转移酶的表达优化包括：（I）优化葡糖醛酸基转移酶的表达， 基于杆状病毒或细菌系统中的苯酚UDPGT; (ii)探索酶的构象特异性， 二氢二醇底物。这些研究预计将增加我们的 了解代谢的分子细节， 环境污染物和药物，并最终提高我们的能力， 来预测新的潜在有毒化合物的代谢情况。

The metabolism and detoxication of potentially toxic xenobiotic molecules relies on the involvement of several membrane-bound enzymes including the cytochromes P-450, microsomal epoxide hydrolase and UDP-glucuronosyltransferase. Epoxide hydrolase catalyzes the trans-addition of water to epoxides that are often generated by the action of the cytochromes P-450 on unsaturated hydrocarbons. UDP-glucuronosyltransferase catalyzes the transfer of the clucuronyl group from UDP-glucuronate to hydrophobic molecules bearing nucleophilic functional groups such as -OH, -SH, -NH2 and - COOH. The net result of these reactions is to enhance the solubility, transport and excretion of the parent molecules. The specific aims for the proposed project period are focused on three general areas of inquiry including the improvement of expression systems for the proteins, the elucidation of the mechanisms of action of the enzymes and preliminary investigations of the structure of the enzymes by X-ray crystallography and biochemical techniques. The specific aims of the project with respect to microsomal epoxide hydrolase are: (I) the elucidation of the kinetic mechanism by pre-steady-state and steady- state techniques; (ii) the definition of the chemical mechanism of catalysis by site-directed mutagenesis and characterization of the ester intermedicate; (iii) evaluation of potential mechanism-based inhibitors of the enzyme; and (iv) production of X-ray diffraction-quality single crystals. The specific aim for the investigations of UDP- glucuronosyltransferase include: (I) optimization of the expression of the phenol UDPGT in a baculovirus-based or bacterial systems and ; (ii) an exploration of the conformer specificity of the enzyme toward dihydrodiol substrates. These studies are anticipated to increase our understanding of the molecular details of the metabolism of environmental pollutants and drugs and eventually enhance our ability to predict metabolic scenarios for new, potentially toxic compounds.